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DRUG TARGETING IN NEOPLASTIC DISEASES Vamshi Krishna .J

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2 Cancer - An Enigma

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3 Cancer is characterized by mutation and deregulation of genes , manifesting itself in the form of unrestrained cell growth. It is principally of two types: - Hematologic – such as leukemia (blood born) - Solid tumors – such as breast cancer Limit less replicative potential Absence of cell repair mechanism Lack of apoptosis Self sufficiency in proliferative growth signals Insensitivity to growth inhibitory signals Characteristics of a canerous cell

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4 Treatment options for cancer Surgery: before 1955 Radiotherapy: 1955-1965 Hyperthermia: 1958-1967 Chemotherapy: after 1965 Immunotherapy and Gene therapy The design of tumor specific delivery of chemotherapeutic agents is a means, to address the issues of the dose-limiting toxic side effects of these agents, by enhancing the fraction of dose actually reaching the tumor while, reducing the amount of drug that reaching the non-targeted organs.

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7 Methods developed to enhance specificity of chemotherapeutic agents DRUG TARGETING A “state – of the – art” technique - First order targeting - Second order targeting - Third order targeting - Passive targeting - Active targeting - Physical targeting

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9 Tumor Physiology & EPR Effect Cancerous growth feeds parasitically on the existing supply of blood and nutrients. As the tumor develops, it can develop it’s own blood vessels but cannot form its own lymphatic system. The blood vessels developed, are often leaky and porous within the tumor interstitium. Interstitial pressure inside the tumor is much higher when compared to the normal cell and viscosity of blood in tumor is much higher –- slower drug migration . Enhanced Permeability and Retention ( EPR ) effect is the property by which certain sizes of molecules (typically liposomes, nanoparticles, and macromolecular drugs) tend to accumulate in tumor tissue much more than they do in normal tissues.

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Large molecules targeted to a tumor, leak out of the porous blood vessels to reach the interstitium. These molecules are then unable to return to the circulation, where as the smaller ones do . Extravasation:

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Pro-drug A pro-drug is a compound resulting from chemical modification of a biologically active compound that will liberate the active form in vivo by enzymatic or hydrolytic cleavage. Modification of the physico-chemical properties to alter the membrane permeability of the parent compound. They exist in inactivated forms until activated by other specific enzymes or biochemicals. They have been taken even further, by including as polymeric pro-drugs in which a drug is covalently linked to a polymeric backbone. E.g.. Mitomycin.

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Simple Soluble Macro Molecular Systems Glycoprotein Gene therapy Monoclonal antibodies Soluble synthetic polymers Prodrug constructs - Challenging factors 1. Requires an activatable version of the drug, and research in its development is comparable in cost to drug development. 2. The technology does not necessarily place the drug where it is needed . This may be improved by a targeting carrier system.

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14 Glycoproteins The family of glycoproteins includes many enzymes, acute phase reactant proteins and plasma proteins. Mannose, galactose and sialic acid are the principle sugars that form the carbohydrate components of this simple macromolecules and tend to confer receptor specificity . Use of glycoproteins as a delivery system for antineoplastic diseases is attractive conceptually due to the targeting specificity afforded by the ligand - receptor interaction principle.

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15 Monoclonal Antibodies By definition the monoclonal antibodies are very specific to their immunological ligands and are thus very appealing drug carriers. The problem of spatial placement is addressed. Monoclonal antibodies have been coupled to cytotoxic anti cancer agents such as doxorubicin and the carrier substrate dextran. The Mabs used in these formulations were directed in theory against epitopes found only on cancer cells . Antibodies tend to be very confirmationally stable and usually retain their binding specificity when combined with other molecules.

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16 Bispecific Antibodies In carcinoma patients the combination treatment of IL-2 and Bis-1F bispecific antibody directed against epithelial glycoprotein -2 and TcR/CD3 complex on T-Lymphocytes elicited an immune response measured by elevated plasma levels of TNF- α and interferon – γ . Through cross linking of T-cell receptor and CD3 complex on the cytotoxic T-lymphocytes and epithelial glycoprotein-2 on the target cell , the lymphocyte tends to be capable of actively lysing the target cells .

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18 Antibody directed enzyme- pro drug therapy (ADEPT ) The need for antibody internalization, which is one of the problems associated with immunoconjugates is a addressed in this strategy, known as Antibody directed enzyme- pro drug therapy (ADEPT). Enzymosomes are liposomal constructs engineered to provide a mini bio environment . Enzymes are covalently immobilized or coupled to the surface of liposomes, therefore, when a non toxic product is administered simultaneously, it is converted by the immobilized enzyme to a potent anti-tumor agent in the vicinity of tumor cell lines.

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19 Immunotoxin conjugate Toxins are molecules that inactivate Viral cytosolic components of the protein synthesis machinery in catalytic manner. Reaching cytosol is the major requirement. Immuno-toxins are the conjugates of antibodies (Mab)/ (Fab) fragments and toxins in which the cell binding moieties of the toxins are replaced by the binding specific chain of Ab.

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Soluble Synthetic Polymer Systems The synthetic polymeric carriers are large enough to avoid filtration and removal by the kidneys but small enough to avoid trapping by the liver and spleen. Many natural and synthetic biodegradable polymers have been investigated as implants, microcapsules, micro particles and nanocapsules in order to achieve prolonged release and targeting of a variety of drugs. Apart from targeting, the backbone of a polymeric carrier molecule provides both controlled, sustained release pattern and a means of protecting the drug form the physiological environment.

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22 Polymer–Drug Conjugates For Intravascular Delivery Release is by hydrolytic or enzymatic cleavage. The drug may be a party of a polymer backbone attached to polymer side chains distributed along the polymer backbone or attached to terminal group of the Polymer backbone. This consists of a water-soluble polymer, a chemotherapeutic agent and a pH–sensitive biodegradable linker.

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Fate of Polymer Conjugate After Intravenous Administration Linker links the polymer and drug in a intact form during its journey through the vascular compartment. Upon reaching the endosomal compartment(vesicular structure after endocytosis) The polymer conjugate is further directed towards lysosomes . P-D conjugates are thus lysosomotropic as they are selectively taken up by them. Due to the acidic environment and the hydrolytic action of lysosomal enzymes, the linker is cleaved. The drug liberated , diffuses across the lysosomal membrane into cytoplasm .The ghost polymer is then released in the blood by exocytosis and is eventually eliminated by the kidneys.

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Fate of Polymer Conjugate After Intravenous Administration

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Design of Polymer Conjugates Polymer : should degrade into non-toxic, non immunogenic, water soluble metabolites that are eliminated easily via renal filtration but be of sufficiently high molecular weight to allow entrapment in the tumor by EPR effect. E.g.: Dextrin (2000-55,000) Poly glutamic acid (30,000 -60,000) Polymer should possess functional groups which are amenable to conjugation with a drug directly or through a linker. Polymer should possess high drug carrying capacity, which in turn results in poor water solubility - Doxorubicin-HPMA conjugates contain about 8%w/w of the drug. - 5-Fluorouracil-Carboxy methyl chitin – 36%w/w of the drug.

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26 Biodegradable polymers Ghost polymer degrades into bio-compatible components and is excreted. Poly( amino acids) are one such class of biodegradable water soluble polymers. Poly-L-glutamic acid is extensively used because of proven non immunogenicity and bio compatibility of its degradation product i.e. L-glutamic acid. E.g. Paclitaxal and Campothecin with poly-L-glutamic acid. Styrene-maleic anhydride–neocarzinostatin (SMANCS) is the only biodegradable polymer studied which limited the bone marrow axis of the drug . The free drug causes severe bone marrow toxicity.

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28 N-(2-hydroxypropyl)methacrylamide copolymer (HPMA) The design of a new cross-linking agent viz., N2,N5-bis ( N-methacryloyl GPLG)Ornithine offered the possibility of use of conjugates with mol.wt. as high as 1230 k Da to maximise the advantage of EPR effect. The invivo cleavage of the cross link by lysosomal enzymes gave rise to polymer fragments below the renal clearance threshold , thus overcoming the accumulation issues.

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Chemotherapeutic agent Extensively studied drug candidates for preparing polymer conjugates are doxorubicin and daunorubicin. The cytotoxic agents seems to be better than the microtubule inhibitors. Since, these can show the effect only after lysis in the cell. The tumor selective accumulation also helps to synergize the radiotherapy when used in conjugation with conjugates of drugs having photosensitizing properties. E.g. Lipiodol paclitaxel Targeting moiety Up take can be enhanced by a tumor receptor – specific ligand . Type of ligand depends on the type of target chosen.

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Incorporation of galactose residues imparts a hepato specific uptake by interacting with the asialglycoprotein receptor present on the hepatocytes. Incorporation of an antibody in the conjugate that recognizes a cell surface antigen specific for the cancer cells. such conjugates containing mAb, CD3, anti EL-4, or antibody fragments or immunoglobulins constitute a subclass called immunoconjugates .

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32 Liposomes

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33 Liposomes

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34 Targeting strategies using liposomes can be designed as: Natural targeting of conventional liposomes. Long circulatory liposomes (Stealth liposomes). Ligand mediated targeting. Use of anti receptor antibodies or antibodies developed against specific surface antigens on the tumor vascular endothelium. Use of stealth liposomes and ligand mediated targeting in combination. Liposomes

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Sterically stabilized liposomes , avoid scavenging through receptor mediated uptake by mononuclear phagocytic cells of reticulo endothelial system rich organs. A fraction of the lipids present, have a polyethylene glycol polymer bound to their head groups. This polymer binds a lot of water creating a water cloud around the liposome, which hides it from the immune system and provide long circulatory behavior . Hence the name, Stealth liposomes. PEG can be substituted by, Sialo-glyco conjugates . - Sialoglycoprotein of human erythrocytes. - Sialoglycopeptide derived fetuin. - Sialic acid conjugated cholesterol substituted pullulan. 35

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The design of liposomes can be such that, they can become leaky a few degrees above the body temperature hence letting the encapsulated material flow out. By tuning the lipid composition to become leaky at a certain temperature above the body temperature, it is possible to heat the tumor locally by either microwave, ultrasound or radio-frequency radiation resulting in a very fast release of the anti-cancer drug, typically a million times faster than from conventional liposomes. 38 Release of drug from liposomes…A Constraint ?

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Liposome targeting to tumors using vitamin and growth factor receptors 39 Advances in liposome technology have resulted in the development of ligand targeted liposomes capable of selectively increasing the efficacy of carried agents against receptor bearing tumor cells. Receptors for vitamins and growth factors have become attractive targets for ligand-directed liposomal therapies due to their high expression levels on various forms of tumor and their ability to internalize after binding to the liposomes conjugated to receptors’ natural ligands (vitamins).

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Triggered release of liposomes 41 An ideal system may involve some time dependent or other specific inducible changes in the liposome membrane or its coating to produce intelligent liposomes that will change their properties (e.g.. leakage rate, fusogenic activity and interaction with particular cells) upon getting specific stimulus following their application. Triggered release signal sensitive liposomes are of following types. - Thermo sensitive liposomes - P H sensitive liposomes - Magneto liposomes - Photo activated liposomes

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42 Dramatic increase in the permeability of liposomes occurred at a temperature (above physiologic temperature).The fluctuations in temperature insitu trigger the release of the contents delivering them at cellular level. In addition, the temperature of the desired site can be artificially manipulated by applying local heating , that could increase the temperature of the desired site and hence concomitant release of the contents. Incorporation of anti target Mab’s on P H sensitive immunoliposomes possess the ability to specifically deliver cytotoxic drugs (methotrexate) to the cytoplasm of the cell .

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43 Immunoliposomes Immunoliposomes are generated by conjugating antibodies either directly to lipid bilayer of liposomes in presence or absence of PEG chains (type I immunoliposomes) or to the distal end of the PEG chain (type II immunoliposomes). Target cell recognition by immunoliposomes is influenced by two factors : - The type of the antibody molecule. - The chemistry of conjugation.

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44 Immunoliposomes

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45 Immunoliposomes, make use of use of hyperthermia or magnetic field for the induced release of it’s contents. Liposomal systems appear to be the most promising carrier systems, for photo sensitizers in the photodynamic therapy of tumors. The photo sensitizer(Eosin) coupled to a phospholipid and incorporated into trinitrophenol(TNP) bearing liposomes, inorder to target the photo sensitizer to B-lymphoma cells , that expresses TNP-specific membrane I g G receptors.

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Niosomes 47 Niosomes are non-ionic surfactant vesicles obtained on hydration of synthetic nonionic surfactants, with or without incorporation of cholesterol or other lipids. They are similar to liposomes but the bilayer in the case of niosomes is made up of non-ionic surface active agents, rather than phospholipids, as seen in the case of liposomes. Liposomes face problems such as; they are expensive, their ingredients like phospholipids are chemically unstable because of their predisposition to oxidative degradation, they require special storage and handling and purity of natural phospholipids is variable.

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Microspheres 48 Many of the biocompatible polymers can be used as small soluble molecular drug carriers. Therapeutic agents can be incorporated into these assembled polymers (particle size less than 200 µ m). Albumin microspheres, Chitosan microspheres and many other synthetic microspheres are being used as carriers for the delivery of several chemotherapeutic agents. Magnetic Microspheres : To minimize the reticuloendothelial clearance and increase the target specificity . They are composed of denatured serum albumin matrix that serves as the vesicle in which anticancer agent and ultrafine particles of magnetite are entrapped.

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49 Definition: Magnetic microspheres are supramolecular particles that are small enough to circulate through capillaries without producing embolic occlusion (<4 μm) but are sufficiently susceptible (ferromagnetic) to be captured in micro vessels and dragged in to the adjacent tissues by magnetic fields of 0.5-0.8 tesla (T)

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NanoParticles 50 The loading of drug into ultrafine colloidal particles in the nanometer size range (10-1000nm)is done, for optimization of drug delivery to the desired site with the either the drug encapsulated, dissolved, adsorbed or covalently attached. Nanoparticles can be prepared using natural hydrophilic polymers ( proteins and polysaccharides ) or synthetic hydrophobic polymers ( PECL, PLA, PLGA, Poly cyanoacrylates, PMMA ). They can entrap various agents in stable and reproducible fashion. Stabilizers such as dextran and its derivatives can be incorporated into nanoparticle surface to modify it’s surface characteristics.

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Implantable Systems 51 Cylindrical monolithic devices of mm or cm dimensions, implanted by a minor surgical incision or injected through a large bore needle into subcutaneous or intra muscular tissue . The drug in implants may be dissolved or dispersed or embedded in a matrix of polymers. Implantable drug-delivery systems can detect chemical signals in the body and release appropriate therapeutic dosages for treatment with the help of biosensors . Improved control of drug levels at the specific site of action is possible, for prolonged duration with significantly small dose. Solid Implants :

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52 In-situ gels consists of biodegradable polymers dissolved in a biocompatible carrier ( DMSO or NMP ). When the liquid polymer system is placed in the body, it solidifies upon contact with aqueous body fluids to form a solid implant. The gel-matrix, thus formed will release the incorporated drug slowly over a period of weeks to months, and ultimately biodegrade depending on the composition used. In-situ Forming Implants :

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Intra-tumoral Drug Delivery 54 The concept of administration of drug directly into the tumor arose from the non uniform and in adequate accumulation of drug or drug carrier in the tumor. Prodrug approach has been successfully utilized for intra tumoral chemotherapy for a variety of drugs. Mitomycin C, conjugated with dextran and subcutaneously implanted in B 16 melanoma, resulted in reduction in tumor growth. It can also be optimized using polymeric implants . Cisplatin-Collagen matrix, BCNU-polyanhydride implant, Vinblastine-Collagen matrix, Methotrexate-Polylactide implant, all have resulted in suppression of tumor.

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55 Chemoembolization Embolization is widely acknowledged form of endovascular therapy. It consists of delivering an embolic material locally through a catheter that has been previously inserted in the vessels supplying the pathological area. Chemoembolization involves the selective arterial embolization of a tumor together with a simultaneous or subsequent local delivery of chemotherapeutic agents. Microcapsule bound intra-arterial chemotherapy has the greatest potential in treating most of the tumors.

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56 Marketed Products

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57 In the recent past, we have witnessed an explosion in research aimed at creating novel drug delivery systems for various kinds of neoplasms. During the next few decades, the clinical evaluation of many of the new tumor targeting drug delivery systems will be in operation. Progress in immunology, human genomics , liposomes & nanoparticle technology etc. should lead to greater insight into the type of targeting molecules that can be used to achieve site-specific drug delivery. As our understanding of the drug action and pathogenesis of different types of neoplasms becomes vivid, more rational approaches to the design of therapeutic systems , that target the tumors with no or reduced side effects, will emerge. Conclusion